Cells must sense their nutritional or environmental conditions and modify their metabolic activity appropriately. Yeast SNF1 (sucrose non-feirmenting) protein kinase and mammalian AMP-activated protein kinase (AMPK) are central components of kinase cascades that act as metabolic sensors of glucose availability and AMP:ATP levels respectively. Protein sequence and functional homology exists between the yeast and mammalian kinase subunits (SNF1/AMPK-xcex1), activation subunits (SNF4/AMPK-xcex3) and the docking subunits (SIP/AMPK-xcex2) that constitute the functional kinase complexes (Hardie, D., et al., Annu. Rev. Biochem. 67:821-55 (1998)).
In yeast, the association of the SNF4 activating subunit with a regulatory region of the SNF1 protein is sensitive to glucose. When glucose concentration is low, the SNF4 protein associates with the regulatory domain of SNF1, and the activity of the catalytic kinase domain is increased, resulting in the derepression of genes required for the metabolism of alternative energy sources. When glucose concentration is high, the SNF1 kinase domain associates with its regulatory domain and kinase activity is inhibited. In mammals, the activation of AMPK, in response to increases in the AMP:ATP ratio, results in the switching on of ATP-producing pathways and the switching off of ATP-consuming pathways. For example, AMPK activation results in the phosphorylation and inactivation of acetyl coenzyme A carboxylase and 3-hydroxy-3-30 methylglutaryl coenzyme A reductase (HMGCoA reductase); but unlike in yeast, the specific functions of the xcex3- and xcex2-subunits are less well defined.
In plants, there is also evidence that carbohydrates control gene expression, growth, metabolism and differentiation. Jang and Shcen, Trend""s in Plant Sciences, 2:208-214 (1997); Koch, Annu. Rev. Plant. Physiol. Mol., 2:509-540 (1996)). An extensivc family of SNF1 homologs and related kinases have been characterized and have been grouped into several subfamilies of SNF1-related kinases (SnRKs) (Halford, et al., Plant Mol. Biol. 37:735-748 (1996)). In addition to exhibiting kinase activity on substrates common to the mammalian and yeast kinases and complementing yeast SNF1 mutants (Alderson, et al. Proc. Natl. Acad. Sci. U.S.A. 88:8602-05 (1991); Muranaka, et al. Mol. Cell Biol., 14:2958-65 (1994)), antisense suppression experiments suggest that plant SNF1 homologs may also be involved in regulation of carbon metabolism in planta. Purcell, et cal., Plant J. 14:195 (1998). However, NPK5,a SNF1 homolog from tobacco was unable to complement an SNF4-deletion yeast mutant strain (xcex94-SNF4), suggesting that NPK5 may require an SNF4-like component for physiological activity in vivo (Muranaka et al., Mol Cell Biol., 14:2958-65 (1994)).
No functional homolog of the SNF4 activating subunit has yet been demonstrated from plants, although a gene sequence (Pv42) isolated from developing bean seeds was reported to have predicted amino acid sequence similarity to SNF4. (Accession No. U40713.) Thus, there exist needs to identify and express plant homologs to yeast SNF4 proteins in order to understand how plants cope with metabolic and strcss conditions in the environment and to modulate these responses to engineer plants resistant to various environmental stresses. In addition, production of genetically engineered plants with improved carbon metabolism and source-sink relationships could be used to improve yields or qualities of harvested plant products. The present invention addresses these and other needs.
The present invention provides SNF4 homologs from plants. In particular, the present invention provides nucleic acid molecules which encode plant SNF4 polypeptides. The polypeptides of the invention comprise an amino acid sequence that has greater than about 70% identity to SEQ ID NO:3.
Also provided is the promoter sequence from SEQ ID NO:2. Promoters of the invention can be operably linked to heterologous nucleic acid sequences and used to drive expression of the heterologous sequences in desired plant tissues.
The present invention further provides SNF1 polypeptides. The SNF1 polypeptides of the invention comprise an amino acid sequence that has greater than about 95% identity to the amino acid sequence of the polypeptide encoded by SEQ ID NO:4. An exemplary SNF1 nucleic acid molecule from tomato is shown in SEQ ID NO:4 (LeSNF1). Preferably, the nucleic acid molecule can specifically hybridize to SEQ ID NO:4 or its complement.
The present invention fuirther provides recombinant expression vectors comprising the nucleic acid sequences of the invention. Preferably, the vectors comprise a plant promoter operably linked to the nucleic acid sequence. The promoter can be either a constitutive promoter, or an inducible promoter.
The present invention also provides for transgenic plants comprising a recombinant expression cassette of the invention. The recombinant expression cassettes are useful in methods of modulating source-sink relationships in plants and thereby enhancing yield or quality of harvested plant products, such as fruit. For example, the nucleic acids of the invention can be used to enhance sink activity and starch or lipid accumulation in seeds. Alternatively, the can be used to enhance sugar accumulation in fruit. The expression cassettes of the invention can also be used to enhance responsiveness to stress conditions in plants.
The term xe2x80x9cstress conditionsxe2x80x9d as used herein generally refers to nutritional and environmental stress that plants encounter in their life cycle. Examples of stress conditions are any nutritional or environmental changes that lead to changes in plant internal metabolic pathways and alterations in the plant""s carbon reserves. Examples of environmental stresses include extreme temperature (e.g. excess heat or cold), high salt, flooding, anoxia, drought, toxic chemicals (e.g. herbicides, heavy metals) and the like.
The term xe2x80x9cplant SNF4 polypeptidexe2x80x9d refers to plant homologs of yeast SNF4. Without wishing to be bound by theory it is believed that the polypeptides of the invention are activating subunits in kinase cascades that act as metabolic sensors of carbohydrate availability and ATP levels in plant cells The proteins of the invention are a component in SNF1 related protein kinases which are composed of kinase subunits (SNF1), activation subunits (SNF4), and docking subunits (SIP). The tenn xe2x80x9cLeSNF4xe2x80x9d refers to plant SNF4 polypeptides derived from tomato (Lycopersicon escuentum).
Plant SNF4 polypeptides of the invention are typically from about 20 amino acids to about 400 amino acids in length, usually from about 100 to about 375, and often from about 200 to about 300 amino acids. A full length plant SNF4 polypeptide of the invention is typically about 375 amino acids.
The term xe2x80x9cplant SNF1 polypeptidexe2x80x9d refers to a plant homolog of the SNF1 subunit of the SNF1-related protein kinase. An example of a SNF1 nucleic acid is the LeSNF1 nucleic acid sequence as shown in SEQ ID NO.:4. An example of a SNF1 amino acid sequence is the LeSNF1 amino acid sequence as shown in SEQ ID NO.:5.
The phrase xe2x80x9cnucleic acid sequencexe2x80x9d refers to a single or double-stranded polymer of deoxyribonucleotide or ribonuclcotidc bases read from the 5xe2x80x2 to the 3 end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.
The term xe2x80x9cpromoterxe2x80x9d refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A xe2x80x9cplant promoterxe2x80x9d is a promoter capable of initiating transcription in plant cells. Such a promoter can be derived from plant genes or from other organisms, such as viruses capable of infecting plant cells.
The term xe2x80x9cplantxe2x80x9d includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endospenn, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.
A polynucleotide sequence is xe2x80x9cheterologous toxe2x80x9d an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety).
A polynucleotide xe2x80x9cexogenous toxe2x80x9d an individual plant is a polynucleotidc which is introduced into the plant by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and include Agrobatcterium-mediated transformation, biolistic methods, electroporation, and the like. Such a plant containing the exogenous nucleic acid is referred to here as a T1 (e.g. in Adrabidopsis by vacuum infiltration) or R0 (for plants regenerated from transformed cells in vitro) generation transgenic plant. Transgenic plants that arise from sexual cross or by selfing are descendants of such a plant.
xe2x80x9cRecombinantxe2x80x9d refers to a human manipulated polynucleotide or a copy or complement of a human manipulated polynucleotide. For instance, a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Clonincg-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley and Sons, Inc. (1994-1998)) of an isolated nucleic acid comprising the expression cassette. In another example, a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second polynucleotide. One of skill will recognize that polynucleotides can be manipulated in many ways and are not limited to the examples above.
Two nucleic acid sequences or polypeptides are said to be xe2x80x9cidenticalxe2x80x9d if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms xe2x80x9cidenticalxe2x80x9d or percent xe2x80x9cidentity,xe2x80x9d in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers and Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
The phrase xe2x80x9csubstantially identical,xe2x80x9d in the context of two nucleic acids or polypeptides, refers to sequences or subsequences that have at least 60%, preferably 70%, more preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity when aligned for maximum correspondence over a comparison window as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.
One of skill in the art will recognize that two polypeptides can also be xe2x80x9csubstantially identicalxe2x80x9d if the two polypeptides are immunologically similar. Thus, overall protein structure may be similar while the primary structure of the two polypeptides display significant variation. Therefore a method to measure whether two polypeptides are substantially identical involves measuring the binding of monoclonal or polyclonal antibodies to each polypeptide. Two polypeptides are substantially identical if the antibodies specific for a first polypeptide bind to a second polypeptide with an affinity of at least one third of the affinity for the first polypeptide.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When usinlg a sequence comparison algonrthm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat""l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (1995 Supplement) (Ausubel)).
Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschuel et al. (1977) Nucleic Acidcs Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechiology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always greater than 0) and N (penalty score for mismatching residues; always less than 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=xe2x88x924, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Nalt. Acad. Sci. USA 89:10915 (1989)).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat""l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.
xe2x80x9cConservatively modified variantsxe2x80x9d applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are xe2x80x9csilent variations, xe2x80x9d which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a xe2x80x9cconservatively modified variantxe2x80x9d where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, e.g., Creighton, Proteins (1984)).
An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
The phrase xe2x80x9cselectively (or specifically) hybridizes toxe2x80x9d refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
The phrase xe2x80x9cstringent hybridization conditionsxe2x80x9d refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biolooyxe2x80x94Hybridization with Nucleic Probes, xe2x80x9cOverview of principles of hybridization and the strategy of nucleic acid assaysxe2x80x9d (1993). Generally, highly stringent conditions are selected to be about 5-10xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Lower stringency conditions are generally selected to be about 15-30 xc2x0 C. below the Tm. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30xc2x0 C. for shoit probes (e.g., 10 to 50 nucleotides) and at least about 60xc2x0 C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as fonnamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 time background hybridization.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
In the present invention, gcnomic DNA or cDNA comprising plant SNF4 or SNF1 nucleic acids of the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here. For the purposes of this disclosure, suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37xc2x0 C., and at least one wash in 0.2xc3x97SSC at a temperature of at least about 50xc2x0 C., usually about 55xc2x0 C. to about 60xc2x0 C., for 20 minutes, or equivalent conditions. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary xe2x80x9cmoderately stringent hybridization conditionsxe2x80x9d include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37xc2x0 C., and a wash in 1X SSC at 45xc2x0 C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
A further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., an RNA gel or DNA gel blot hybridization analysis.